The conventional therapies like surgery and radiation are effective in eliminating large volume tumors but it is not practical to employ these approaches for treating the systemic residual microscopic malignancy. Chemotherapy may be effective in inhibiting or eliminating the systemic residual microscopic malignancy but recurrence eventually occurs due to the development of drug resistance. Delivery of cytokine gene, like IL-12, via electroporation not only eradicates residual tumors but also prevents tumor recurrence. However, monocytokine electroporaiton gene therapy generally only eliminates in the less aggressive tumors. For example, neither electroporation IL-12 nor other cytokine genes alone can eradicate the highly malignant 4T1 breast adenocarcinoma via systemic administration. To eradicate the highly malignant tumors, multiple systemic approaches have been tested using our unique electroporation-based gene delivery and gene expression systems during the current award period. We discovered that sequential systemic expression of IL-12 and IL-27 eradicates not only the less aggressive CT26 colon tumors but also highly malignant 4T1 breast tumors. The cured mice reject rechallenged homologous tumor cells in 83% of mice. Intriguingly, this potent therapeutic efficacy cannot be achieved by both IL-12 and IL-27 sequential gene therapy in a reversed order of administration and co-administration of IL-12 and IL-27. Therefore, one of goals in this application is to elucidate why IL-27 electroporation gene therapy is required after IL-12 electroporation gene therapy for eradicating tumors. We also found that doxorubicin, an anti-neoplasm drug, potentiates IL-12- mediated eradication of highly malignant 4T1 tumors and induces the accumulation of the toxicityinducing cytokine IFN into tumors but not in the normal tissues. Becasue both Dox plus IL-12 and IL- 12/IL27 sequential gene therapy independently induces eradication of this highly malignant 4T1 tumors from ~30% mice, it is likely a combination of both will further improve the theraputic efficacy. Because the IL-12-induced toxicity-inducing cytokine IFN will be accumulated in tumors by Dox treatment, inclusion of Dox in the sequential IL-12 and IL-27 electroporation gene therapy may increase the safety. Therefore, the applicant will determine the therapeutic efficacy and the safety of the sequential systemic IL-12 and IL-27 electroporation gene therapy in combination with Dox treatment. A positive result will support the central hypothesis that the sequential systemic IL-12 and IL-27 gene therapy in combination with Dox is an effective approach for eliminating residual microscopic malignancy and will reduce the systemic IL-12 treatment-caused toxicity.